Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator

  1. Takahisa Miyao
  2. Maki Miyauchi
  3. S Thomas Kelly
  4. Tommy W Terooatea
  5. Tatsuya Ishikawa
  6. Eugene Oh
  7. Sotaro Hirai
  8. Kenta Horie
  9. Yuki Takakura
  10. Houko Ohki
  11. Mio Hayama
  12. Yuya Maruyama
  13. Takao Seki
  14. Hiroto Ishii
  15. Haruka Yabukami
  16. Masaki Yoshida
  17. Azusa Inoue
  18. Asako Sakaue-Sawano
  19. Atsushi Miyawaki
  20. Masafumi Muratani
  21. Aki Minoda
  22. Nobuko Akiyama
  23. Taishin Akiyama

  • Curated by eLife

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    Evaluation Summary:

    The present work by Miyao and Miyauchi et al. provides new insights into the heterogeneity of mTEC. With single-cell approaches and the Fucci2a mouse model, they have found a proliferating mTEC sub-population that may be a precursor of mature mTECs expressing the Aire gene. The findings are potentially important for the field.

    (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. The reviewers remained anonymous to the authors.)

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Abstract

Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by the transcriptional regulator, AIRE. Whereas AIRE-expressing (Aire + ) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase-accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules, CD80 (Aire + CD80 hi ). Proliferating Aire + CD80 hi mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire + CD80 hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire + mTEC precursors during the Aire + mTEC differentiation process of the postnatal thymus.

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  1. Version published to 10.7554/elife.73998 on eLife
  2. eLife

    Evaluation Summary:

    The present work by Miyao and Miyauchi et al. provides new insights into the heterogeneity of mTEC. With single-cell approaches and the Fucci2a mouse model, they have found a proliferating mTEC sub-population that may be a precursor of mature mTECs expressing the Aire gene. The findings are potentially important for the field.

    (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. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Using the combinatorial interpretation of single cell-based technology, the authors attempted to characterize differentiation dynamism of heterogenous mTEC clusters.

    By analyzing the data, they found that Aire+ transit-amplifying TECs (TA-TECs) using Fucci system. In addition, they showed that TA-TECs could potentially differentiate into TSA-expressing functional mTEC. TA-mTECs appear to be maintained in the thymus from the youth to the adulthood.

    Although they produced many datasets at single-cell resolution, the conclusion did not fully exploit the strength of integrative analysis of the transcriptome and chromatin accessibility. It is possible that the authors could obtain the same conclusion using the already existing datasets or using the data from either RNA-seq or ATAC-seq because their dataset itself was not different from the ones in many previous publications, as they mentioned in the manuscript.

    Nevertheless, the high-quality data of single cell-based technology in the present study must be valuable for researchers in the field. Their finding of "transit-amplifying Aire+ TSA-low mTECs as a potential precursor population of functional mTECs" can contribute to our understanding of the mTEC development mechanism that plays a key role in immune tolerance.

  4. eLife

    Reviewer #2 (Public Review):

    The paper is a careful and solid analysis to identify the cycling precursor cell for Aire-positive medullary thymic epithelial cells (mTECs) in the postnatal thymus. The study uses an extensive set of single-cell analyses and importantly, the Fucci2a mouse model, which enables the identification of exact transit-amplifying cell type. Overall this is an important study as it shows the differentiation dynamics and heterogeneity of mTECs in the adult thymus.

    1. The scATACseq experiment included a relatively low number (n=2) of mice. Understandably this is a complicated experiment, however, the variation between individual animals remains unknown and may influence the interpretation of the results. The authors are advised to show the clustering analysis for the two animals separately in a supplement to confirm that the changes are seen in both animals, not just in one. Similarly, the individual scRNA-seq UMAPs of the three animals should be included in the supplement. The authors should discuss the limitations of small groups in single-cell experiments in the Discussion. Technically it is positive to see the integration of scATAC and scRNA seq results.

    2. Genes expressed in clusters should be provided in the supplement. Without this information, it is difficult to decide about the cluster annotations and to compare them with other similar studies. The authors should give an unbiased list of all genes expressed in mTECs, in particular those genes that are expressed in TACs. To harmonize the findings in the field, it would be useful for the readers if the authors would compare their cluster-specific genes for the overlap with TEC single-cell clusters from other studies (Bornstein et al 2018, Dhalla et al 2020, Baran-Gale et al 2020, Wells et al 2020).

    3. As a proliferating cell type, TACs should express multiple genes associated with cell cycling. The authors focus on Mki67 only but did R1 TAC express other proliferating cell markers, which would support the claim that these are indeed actively dividing cells?

    4. The candidate cell population for TACs, cluster R1 expresses Aire and the proliferating cell marker Mki67. R1 also expresses Ccl21a. The authors did subclustering of R1 and found that R1A-D express Aire whereas R1E has a higher expression of Ccl21a. The authors note that "Thus, it is possible that TECs expressing cell-cycle-related genes, proposed by scRNA-seq analysis, contain at least two proliferating TECs subsets having different chromatin accessibilities and gene expression profiles." To confirm that both R1A-D and R1E subsets are proliferating TACs, the authors might show the proliferating gene markers in these subsets. Was Mki67 expressed among all R1 subpopulations? Would this argue for the presence of TAC among both Aire+ and Aire- cell populations?

    5. Wells et al 2020 (ELife) recently reported mTEC TACs to be mTEClo preceding Aire expression and giving rise to both Aire+ and Ccl21a+ cells. How do the authors reconcile their results (TAC as Aire+) with Wells et al paper (TACs as Aire-)? How to interpret the finding that Wells et al find Ki67 significantly higher in mTEClo than in mTEChi, implying that cell division precedes high the expression of Aire?

    6. In RTOC experiment adult mCherry-lo cells are transferred into embryonic thymic organ culture where they turn mCherry-hi (Figure 6A). The authors show this to confirm the differentiation of mCherry-lo to mCherry-hi. This is indeed a strong support for their conclusion, but would the authors agree that transferring adult mTECs into the embryonic cell environment may trigger other signaling pathways that induce the upregulation of the cell cycle and mCherry expression? Would the same happen if they would transfer these cells to the adult thymus?

  5. eLife

    Reviewer #3 (Public Review):

    This study carefully addresses the questions raised in the introduction and provides convincing evidence:
    - confirming the existence of a cycling mTEC population expressing Aire and clustering between/ next to mTEChi amd mTEClo.
    - showing that this cycling population is sustained by a pattern of chromatin accessibility different from those corresponding to mTEChi or mTEClo.
    - demonstrating that the cycling CD80high mTECs that the authors isolate are TACs giving rise to mature mTEChi and post-Aire mTECs in RTOC systems, thereby validating ex vivo the previously known in silico-inferred trajectory of cycling mTECs.

    Using "revealed" in the title may be misleading, letting think that it's the first time that the cycling mTEC population expressing Aire is identified by single-cell approaches (Wells 2020, Dhalla 2020, Baran-Gale 2020).

    The conclusions of the paper are mostly well supported by the data. However, some points need clarification:

    1. The authors nicely confirm, by the fine analysis of their scRNA-seq data, that the TAC pop contains Aire+ cells and Ccl21+ cells in a visually mutually exclusive manner. However, they don't formally clarify whether the Ccl21-expressing TACs have differences in their chromatin accessibility pattern compared to the Aire-expressing TACs.
      It's also worth showing the expression of CD80 in the scRNA-seq UMAP of TACs alone and in the one of all TECs (Fig2). This would notably allow to determine whether CD80 expression is restricted to Aire-positive TACs or encompasses Aire-negative TACs (Ccl21+).

    2. Trajectory analyses provide a nice confirmation of published results identifying a trajectory from TACs to mTEChi. However, the authors don't discuss whether their data support a potential trajectory from TACs to mTEClo (already suggested/ reported) which seems to be present in Fig 3-fig sup 2B. Would this mean that some TACs could mature into Ccl21+ mTECs (mTEClo)? and if so, how Aire and Ccl21 are expressed in these TACs? Which TAC sub-cluster do they belong to?

    3. The authors focus their study on the CD80high cycling cells (Aire+). Figure 4 show transcription profiles of the isolated cycling CD80+ mTECs. Dots in the "TSA genes" panel (right) don't appear in the "All genes" panel (left). Are those lost dots of TSA genes?
      In Fig 4E (left) low expressed genes seem to be skewed towards Venus- mTEChi (in comparison to high expressed genes). A statistical assessment for the comparisons of the TSA, Aire-dep TSA and Aire-indep TSA profiles to the general profile (Fig 4E and 4G), considering expression levels, would comfort the visual assessment.

    4. In Fig4F, Aire expression is similar between Venus+ and Venus-. Is it compatible with Fig 4A showing less Aire-expressing cells in Venus+ than in Venus-?

  6. Version published to 10.1101/2021.10.04.463004v1 on bioRxiv