Fig. 3: Age-associated TECs are derived from FOXN1-expressing cells.

a, Representative flow cytometry plots from 12–18-mo male and female Foxn1^nTnG mice at the indicated ages, gated on tdTom⁻GFP⁺ cells. tdTom⁻GFP⁺EpCAM⁺ cells were then assessed for expression of the conventional TEC markers UEA1 and Ly51. b, Claudin-3 and UEA1 expression on tdTom⁻GFP⁺EpCAM⁺ cells in Foxn1^nTnG mice, and quantification of claudin-3 on UEA1⁺ mTEC and UEA1⁻Ly51⁻ DN-TECs (n = 4 biological replicates representing individual mice). c, Podoplanin (Pdpn) and PDGFRa expression on tdTom⁻GFP⁺EpCAM⁻ cells (n = 4 biological replicates representing individual mice). d, Number of GFP⁺EpCAM⁺UEA1⁻Ly51⁻Cldn3⁺ aaTEC1 and GFP⁺EpCAM⁻PDPN⁺PDGFRα⁻ aaTEC2 cells in 2-mo (n = 6) and 18-mo (n = 4) mice. e, scRNA-seq was performed on CD45⁻ cells isolated from male and female 20-mo Foxn1^tdTom and age-matched WT mice, and integrated into the epithelial data described in Fig. 1c–e. UMAP of 8,505 cells of the epithelial compartment in the integrated data showing the TEC annotated subsets (top) and overlaid expression of tdTomato (bottom). Scale represents log-transformed average expression of the tdTomato-WPRE element. f, RNA velocity on selected TEC populations in 2-mo (top) or 18-mo (bottom) mice. n_2mo = 1,989; n_18mo = 3,382. g, Vein plots describing the continuous transition of 18-mo early^prog, mTEC1, mTEC^prol and aaTEC subsets to their predicted descendants (represented by diagonal flows) and the dynamic relative frequencies (vein width on the y axis) of these TEC subsets in the thymus over the binned pseudotime. h, Expression of thymocyte markers Thy1 and Lck overlaid on the 18-mo spatial transcriptomics dataset. Outline represents thymocyte-poor area overlaid onto heatmap showing aaTEC1 or aaTEC2 signatures. i, Two representative images in 12–18-mo male and female Foxn1^nTnG mice showing tdTomato and GFP expression with HD-TEC areas highlighted, with few or no tdTomato⁺ cells. Scale bar, 50 μm. j, Human tissue sections from a 50-year-old woman. Shown are consecutive sections with H&E, cytokeratin or CD1a staining. k, aaTEC1 and aaTEC2 gene signatures (top 20 marker genes from our mouse data converted to human orthologs; Supplementary Fig. 3 and Supplementary Table 3) were overlaid on human thymic epithelial cells (n_TEC = 40,144) from single-cell sequencing datasets generated and published elsewhere^9,19,20. Summary data represents mean ± s.e.m. and each dot represents an individual biological replicate. Statistics were generated (b–d) using a two-tailed Mann–Whitney test.