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Thymic alveolar type 2 epithelial mimetic cells revealed by RUNX1 deficiency

Nature Immunology (2026) Cite this article

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Abstract

Autoimmune diseases can affect the lungs, and the mechanisms used to prevent autoimmune lung disease are incompletely understood. Recent studies in the thymus have identified unique populations of medullary thymic epithelial cells (mTECs) called mimetic cells that transcriptionally mimic peripheral epithelial populations. These mimetic cells have important functions in the thymus in immune tolerance. Here, we used a mouse line with thymic-specific deletion of Runt-related transcription factor 1 (Runx1) to identify a new mimetic cell akin to alveolar type 2 (AT2) lung epithelial cells. AT2 mTECs express surfactant genes, and with Runx1 deletion AT2 mTECs expand, resulting in a loss of AIRE+ mTECs and defects in T cell selection. RUNX1 suppresses AT2 mTECs by inhibiting epidermal growth factor receptor signaling. Together, our results identify a thymic mimetic cell type that mimics lung AT2 epithelial cells, further uncovering unrealized epithelial diversity in the thymus.

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Fig. 1: Runx1 deletion results in a loss of AIRE+ mTECs.
The alternative text for this image may have been generated using AI.
Fig. 2: AT2 mTECs revealed by Runx1 deletion.
The alternative text for this image may have been generated using AI.
Fig. 3: Runx1 deletion results in an expansion of SFTPC-expressing mTECs.
The alternative text for this image may have been generated using AI.
Fig. 4: Runx1 deletion results in alterations to thymic lymphocyte populations.
The alternative text for this image may have been generated using AI.
Fig. 5: Thymic surfactant gene expression prevents surfactant autoantibodies.
The alternative text for this image may have been generated using AI.
Fig. 6: RUNX1 binds to EGFR signaling pathway genes.
The alternative text for this image may have been generated using AI.
Fig. 7: EGF promotes SFTPC+ mTEC development.
The alternative text for this image may have been generated using AI.

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Data availability

All newly generated genomics data, including CUT&RUN data and scRNA-seq data, have been deposited at the NCBI GEO Database and are available under accession numbers GSE290143 (CUT&RUN) and GSE290144 (scRNA-seq). Previously reported data are available from GEO under accession numbers GSE226493, GSE1460657, GSE144877, GSE194253, GSE147520, GSE267785 and GSE140654. The PhIP–seq data are available at the Dryad Digital Repository under accession IDs Q66H4FM2 and Q6BG2M7B. Source data are provided with this paper.

Code availability

All code is available at GitHub at https://github.com/j-germino/Runx1-integration and https://github.com/jhsin/scRNA-seq-re-analysis-of-aged-thymus.

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Acknowledgements

Flow cytometry was performed at the UCSF Parnassus Flow Core, and high-throughput sequencing was performed at the UCSF Functional Genomics Core Facility. This work was supported by NIH grant R01AI165829 (to M.R.W.), a UCSF REAC award (M.R.W.) and the UCSF Parnassus Flow Core (RRID:SCR_018206, supported by NIH P30DK063720).

Author information

Authors and Affiliations

  1. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Jun Hyung Sin, Joe Germino, Juliana Sucharov, Xian Liu & Michael R. Waterfield

  2. Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA

    Jun Hyung Sin, Sloan H. Phillips, Aaron Bodansky & Michael R. Waterfield

  3. Department of Pathology, University of California, San Francisco, San Francisco, CA, USA

    Christopher J. Bowman

  4. Diabetes Center, University of California, San Francisco, San Francisco, CA, USA

    Joe Germino, Xian Liu, Yi Wang, Corey N. Miller, Audrey V. Parent & Mark S. Anderson

  5. 10x Genomics, Pleasanton, CA, USA

    Yi Wang

  6. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Mark S. Anderson

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  1. Jun Hyung Sin
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Contributions

J.H.S. and M.R.W. conceived the study, performed experiments, analyzed data and wrote the manuscript. C.J.B. assisted with CUT&RUN. J.G. assisted with scRNA-seq data analysis. S.H.P., J.S. and M.S.A. assisted with data analysis and manuscript preparation. A.B. assisted with PhIP–seq analysis. X.L., C.N.M. and Y.W. assisted with AIRE-deficient scRNA-seq. A.V.P. assisted with human scRNA-seq analysis.

Corresponding author

Correspondence to Michael R. Waterfield.

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Y.W. is employed by 10x Genomics. The other authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: L. A. Dempsey, in collaboration with the rest of the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Runx1 transcript is expressed in MHC-IIlow mTECs.

a, Relative expression of Runx1 mRNA in stromal cells. Downloaded from the Immgen expression database. b, qPCR of Runx1 gene expression in sorted MHC-IIhigh and MHC-IIlow mTECs and Ly51+ cTECs from Foxn1-Cre (-)/Runx1fl/fl (Cre(-)) and Foxn1-Cre(+)/Runx1fl/fl (Cre(+)) mice. c, qPCR of Runx1 gene expression in sorted single positive (SP) CD4+ thymocytes, SP CD8+ thymocytes, double positive (DP) CD4+/CD8+ thymocytes, splenic CD4+ T cells, splenic CD8+ T cells, and splenic CD19+ B cells. d, Confocal microscopy of cytokeratin 5 (KRT5) (red) and cytokeratin 8 (KRT8) (green) from 30-day old thymi. Images taken at 63X magnification. Scale bars = 5 μm. Data are representative of three independent experiments. e, Flow cytometry (left) and percentage (right) of MHC-IIhigh cTECs gated on CD11c−/CD45−/EpCAM+/Ly51+/UEA-1−/IAb+ TECs (n = 5 mice; two independent experiments). f, Flow cytometry (left) and percentage (right) of Ki67+ cTECs gated on CD11c−/CD45−/EpCAM+/Ly51+/Ki67+ TECs (n = 5 mice; two independent experiments). g, Flow cytometry and percentage/absolute number of positively selected T cells gated on TCRβ and CD69 (n = 3 Cre(-) and n = 5 Cre(+) mice; two independent experiments). (e-g) In graphs, the bars correspond to the mean, with error bars showing ±SD of values shown, and each data point represents an individual mouse. (e-g) P values by unpaired two-tailed t-test.

Source data

Extended Data Fig. 2 Runx1-deletion in TECs does not alter thymic tuft cells.

a, Confocal microscopy of cytokeratin 5 (KRT5) (red), Aire (green), and DAPI (blue) of 30-day old thymi from Foxn1-Cre (-)/Runx1fl/fl (Cre(-)) and Foxn1-Cre(+)/Runx1fl/fl (Cre(+)) mice. Images taken at 20X magnification and scale bar = 25 μm. Inserts taken at 200X magnification and scale bar 5 μm. b-c, Flow cytometry (left) and percentage and absolute number (right) of Ki67+ mTECs gated on CD11c−/CD45−/EpCAM+/Ly51−/IAb+ TECs (b) or Ki67+/Aire+ mTECs gated on CD11c−/CD45−/EpCAM+/Ly51−/IAb+/Aire+ mTECs (c) (n = 6 mice; two independent experiments). d, Flow cytometry (left) and percentage and absolute number (right) of Apotracker+ mTECs gated on CD11c−/CD45−/EpCAM+/Ly51−/IAb+ mTECs (n = 4 mice; two independent experiments). e-f, Flow cytometry (left) and percentage and absolute number (right) of DCLK1+ thymic tufts cells (e), L1CAM+ thymic tufts cells (f) gated on CD11c−/CD45−/EpCAM+/Ly51−/IAb+ TECs (n = 3 (e) and n = 4 (f) mice; (e) representative experiment, and (f) two independent experiments). g, Confocal microscopy of cytokeratin 10 (KRT10) (red), DCLK1 (green), and DAPI (blue) of 30-day old thymi. Images taken at 20X magnification. Scale bars = 25 μm. (b-f) In graphs, the bars correspond to the mean, with error bars showing ±SD of values shown, and each data point represents an individual mouse. (b-f) P values by unpaired two-tailed t-test. (a,g) Data are representative of three independent experiments.

Source data

Extended Data Fig. 3 RUNX1 is expressed in human mTECs.

a-b, UMAPs (a) and violin plots (b) of marker genes from scRNA-seq data from sorted CD11c−/EpCAM+/Ly51− mTECs. c-d, scRNA-seq of human thymic epithelial cells. UMAP of human mTEC clusters and RUNX1 expression (c). Violin plot of RUNX1 expression (d). e, UMAP of scRNA-seq of MHC-IIlow mTECs from C57BL/6 mice merged with scRNA-seq from Foxn1-Cre(+)/Runx1+/+ mTECs to identify AT2 mTEC. Red numbers are the percentage of AT2 mTECs.

Extended Data Fig. 4 scRNA-seq analysis of Lung AT2 and AT2 mTECs.

a, UMAPs of cell distributions from lung scRNA-seq and Foxn1-Cre(+)/Runx1fl/fl mTECs scRNA-seq. b-c, Heatmap (b) and UMAPs (c) showing characteristic marker genes from populations in (a). d-e, Metascape analysis of differentially expressed genes between AT2 mTEC and Lung AT2.

Extended Data Fig. 5 Runx1-deletion results in enhanced surfactant gene expression.

a-d, Volcano plots of DGE from scRNA-seq analysis of Foxn1-Cre(+)/Runx1+/+ (WT) mice and Foxn1-Cre(+)/Runx1fl/fl (KO) mice for Ccl21alow (a), Late Aire (b), Keratinocyte-like mTECs (c), and M cell mTECs (d). e, Confocal images of SFTPC (green), DCLK1 (red), and DAPI (blue) from the thymus of Foxn1-Cre (-)/Runx1fl/fl (Cre(-)) and Foxn1-Cre(+)/Runx1fl/fl (Cre(+)) mice. Images taken at 63X magnification. Scale bars = 25 μm. f, Confocal images of SFTPC (green), KRT10 (red), and DAPI (blue) from the thymus of Cre(-) and Cre(+) mice. Images taken at 190X magnification. Scale bars = 15 μm. g-i, Violin plots of chemokine gene expression from scRNA-seq. (a-d) Differentially expressed genes in volcano plots were determined using a two-sided Wilcoxon rank-sum test. (e,f) Data are representative of three independent experiments.

Extended Data Fig. 6 Runx1-deletion in TECs does not alter SP or DP thymocyte populations.

a, Flow cytometry and percentage/absolute number of thymic single positive (SP) CD4+ T cells, SP CD8+ T cells, double positive (DP) T cells, and double-negative (DN) T cells from 30-day-old Foxn1-Cre(-)/Runx1fl/fl mice (Cre(-)) and Foxn1-Cre(+)/Runx1fl/fl mice (Cre(+)) (n = 8 mice; two independent experiments). b, Flow cytometry of B220−/Nk1.1−/TCRγδ−/CD25−/CD5+/TCRβ+ signaled and B220−/Nk1.1−/TCRγδ−/CD25−/CD5−/TCRβ− non-signaled T cells Cre(-), Cre(+), and Foxn1-Cre(+)/Ikzf1fl/fl (Ikzf1fl/fl) mice (n = 3 Cre(-), n = 5 Cre(+), and n = 2 Ikzf1fl/fl mice; two independent experiments). c, H&E staining of the lacrimal gland, and salivary gland. Scale bars = 500 μm. Bar graphs quantitate lymphocytic infiltrate (n = 5 Cre(-) and n = 4 Cre(+) mice). d, Merged UMAP of scRNA-seq data from Foxn1-Cre(+)/Runx1fl/fl (Cre(+)) mTECs, Aire WT mTECs, and Aire−/− mTECs. Approximately 12,000 mTECs were sequenced for both the Aire WT and Aire−/− datasets. e, Overlay of cells from each genotype on the merged UMAP. Red numbers represent the percentage of AT2 mTECs in each genotype. f, Bar graphs representing the percentage of mimetic cells found in Aire WT vs Aire−/− mTECs. (a-c) In graphs, the bars correspond to the mean, with error bars showing ±SD of values shown, and each data point represents an individual mouse. (a-b) P values by unpaired two-tailed t-test. (c) P value by two-tailed nonparametric Mann Whitney.

Source data

Extended Data Fig. 7 Runx1 suppresses EGFR-induced genes in TECs.

a, Location of Runx1 binding sites relative to the transcriptional start site (TSS) from GREAT (Genomic Regions Enrichment of Annotations Tool). b, Motif analysis of Runx1 binding sites from CUT&RUN using MEME-ChIP (Motif Analysis of Large Nucleotide Datasets). The top 5 motifs identified in CUT&RUN were Runx motifs. c, Metascape analysis of genes increased in Runx1-deficient mTECs from scRNA-seq DGE expression comparing total mTECs (all mTEC clusters combined) from Foxn1-Cre(+)/Runx1+/+ (WT) mice versus Foxn1-Cre(+)/Runx1fl/fl (KO) mice. d, Overlay of EGFR-induced genes on scRNA-seq DGE expression comparing total mTECs (all mTEC clusters combined) in WT mice versus KO mice. Blue numbers represent the number of EGFR induced genes in WT mTECs and red numbers represent the number of EGFR induced genes in KO mTECs. e, Overlay of EGFR-induced genes from specific timepoints on scRNA-seq DGE expression comparing total mTECs from WT mice versus KO mice. Blue numbers represent the number of EGFR induced genes in WT mTECs and red numbers represent the number of EGFR induced genes in KO mTECs at each of the indicated timepoints. f-h, Merged genome browser tracks for duplicate CUT&RUN data for IgG, Runx1, and H3K27ac at growth factor receptor gene loci. (d-e) Differentially expressed genes in volcano plots were determined using a two-sided Wilcoxon rank-sum test.

Extended Data Fig. 8 Runx1 binds to ERK-MAPK, mTOR and JAK-STAT gene loci.

a, Merged genome browser tracks for duplicate CUT&RUN data for IgG, Runx1, and H3K27ac at ERK-MAPK gene loci. b, Heatmap of gene expression for total mTECs (all mTEC clusters combined) from Foxn1-Cre(+)/Runx1+/+ (WT) mice versus Foxn1-Cre(+)/Runx1fl/fl (KO) mice for select ERK-MAPK genes in (a). c, Merged genome browser tracks for duplicate CUT&RUN data for IgG, Runx1, and H3K27ac at gene loci for negative regulators of mTOR. d, Heatmap of gene expression for total mTECs (all mTEC clusters combined) from Foxn1-Cre(+)/Runx1+/+ (WT) mice versus Foxn1-Cre(+)/Runx1fl/fl (KO) mice for negative regulators of mTOR and PI3K. e, Merged genome browser tracks for duplicate CUT&RUN data for IgG, Runx1, and H3K27ac at JAK-STAT gene loci. f, Merged genome browser tracks for duplicate CUT&RUN data for IgG, Runx1, and H3K27ac at gene loci for negative regulators of JAK-STAT. g, Heatmap of gene expression for total mTECs (all mTEC clusters combined) from Foxn1-Cre(+)/Runx1+/+ (WT) mice versus Foxn1-Cre(+)/Runx1fl/fl (KO) mice for positive regulators (Jak1/Il6st) and negative regulators of JAK-STAT (Pias/Socs/Ptpn).

Extended Data Fig. 9 Thymic growth factor and growth factor receptor expression.

a, UMAP of thymic CD45− cells (epithelial, mesenchymal, endothelial) from multiple timepoints (Day 3, Day 7, Day 14) of neonatal development. b, Heatmap of marker gene expression for epithelial, mesenchymal, and endothelial populations. c, UMAPs of marker genes for epithelial, mesenchymal, and endothelial populations. d-g, UMAPs and average gene expression for (d) Igf1r, (e) Fgfr2, (f) Igf2, and (g) Fgf10 in cTECs, mTECs, fibroblasts, and endothelial cells.

Extended Data Fig. 10 AT2 mTECs expand in juvenile Foxn1-Cre(+)/Runx1fl/fl/CBG mice and can develop through an Aire-expressing stage or independently of Aire.

a-c, Flow cytometry and percentage of GFP+ mTECs from Foxn1-Cre(+)/Runx1fl/fl/CBG mice (Cre(+)/CBG) at the indicated timepoints (n = 2 (Day 1), n = 2 (Day 4), n = 4 (Day 7), n = 4 (Day 30), and n = 2 (Day 140); two independent experiments). d-f, Flow cytometry and percentage of MHC-IIhigh and MHC-Illow mTECs in Foxn1-Cre(-)/Runx1fl/fl/CBG mice (Cre(-)/CBG) mTECs and Foxn1-Cre(+)/Runx1fl/fl/CBG mice (Cre(+)/CBG) mice at the indicated timepoints. g, IGV CUT&RUN tracks for the indicated genes. h, Cre(-)/CBG mice were treated with EGF for either two or three weeks and flow cytometry was performed for GFPlow and GFPhigh AT2 mTEC (n = 5 mice treated with vehicle for 2 weeks, n = 5 mice treated with EGF for 2 weeks, n = 2 mice treated with vehicle for 3 weeks, and n = 3 mice treated with EGF for 3 weeks; two independent experiments). i, Flow cytometry (left) and percentage and absolute number (right) of GFP+ mTECs from 30-day-old Cre(-)/CBG and Foxn1-Cre(+)/Runx1+/fl/CBG mice (Het/CBG) 21 days after irradiation and treated with EGF or Vehicle for three weeks (n = 1 vehicle-treated Cre(-)/CBG, n = 2 EGF-treated Cre(-)/CBG, n = 1 vehicle-treated Het/CBG, and n = 1 EGF-treated Het/CBG mice; representative experiment). j, Cre(-)/CBG mice were treated with EGF for three weeks and qPCR of AT2 marker genes was performed on sort purified GFPlow, GFPhigh, and a control population (GFPneg) (n = 3 mice; representative experiment). k, qPCR of Runx1 gene expression from sorted MHC-IIlow mTECs from Foxn1-Cre(-)/Runx1fl/fl mice (Cre(-)/Runx1fl/fl), Foxn1-Cre(+)/Runx1+/fl mice (Cre(+)/Runx1+/fl), and Foxn1-Cre(+)/Runx1fl/fl mice (Cre(+)/Runx1fl/fl) (n = 4 Cre(-)/Runx1fl/fl, n = 2 Cre(+)/Runx1+/fl, and n = 2 Cre(+)/Runx1fl/fl mice; representative experiment). l-m, Flow cytometric analysis of CD11c−/CD45−/EpCAM+/Ly51−/tdtTmt+/GFP+ mTECs from (l) Aire-CreERT2/R26tdTomato/ Runx1+/fl (CBG(-)) mice or (m) Aire-CreERT2/R26tdTomato/Runx1+/fl/ CBG (CBG(+)) mice treated with tamoxifen and EGF for two weeks (lineage trace). n, Flow cytometry (left) and percentage and absolute number (right) of GFP+ mTECs from 30-day-old Het/CBG mice treated with IGF2 or Vehicle for two weeks (n = 2 mice; representative experiment). (h,j) In graphs, the bars correspond to the mean, with error bars showing ±SD of values shown, and each data point represents an individual mouse. (h,j) Statistical significance was calculated using a one-way ANOVA, and grouped comparisons were corrected using Tukey’s multiple comparison test.

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Sin, J.H., Bowman, C.J., Germino, J. et al. Thymic alveolar type 2 epithelial mimetic cells revealed by RUNX1 deficiency. Nat Immunol (2026). https://doi.org/10.1038/s41590-026-02536-0

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