Figure 3.

Thymic SCs reside within the subcapsular and perivascular niches and express BCAM

(A) 3D reconstruction of thick sections (300 μM) of human postnatal thymus stained with fibronectin (FN1, green). FN1 stains highly dense tubular structures in the medulla (M) and subcapsular (SCap) regions in the cortex (C). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 200 μm.

(B) Immunofluorescence labeling of thymic epithelial cells co-stained with anti-ITGA6 (CD49f) antibody (magenta), FN1 (green), and EpCAM (gray). Left: co-staining in the subcapsular region; right: co-staining in the medullary area. Asterisks (^∗) indicate areas of colocalization. Nuclei counterstained with DAPI. Scale bars, 50 μm (n = 4, human thymi).

(C) Top: representative immunofluorescence image of an entire human thymus paraffin section (5 μm) analyzed for spatial transcriptomics (n = 4). FN1 staining is shown in green, CD45 and CD3 in red, while KRT18 areas in cyan. Nuclei counterstained with SYTO85 dye. Regions of interest (ROIs) localization is displayed and named in white for each type. Scale bars, 5 mm. Bottom: representative image of each ROI segmented for KRT18-positive cells is displayed in white and labeled as follows: HB (Hassall’s body), FN1 medulla (FNm), central cortex (CC), and subcapsular (SCap).

(D) Dimension reduction analysis displays transcriptional variation of ROIs along PCA2 versus PCA3. The heatmap graph shows the gene expression of PolyKRT and specialized cortical and medullary signature per each ROI, HB (dark red), FNm (red), CC (dark green), and SCap (green). Scale log₁₀ (−3,3).

(E) UMAP feature view plot of BCAM showing the expression in the PolyKRT cluster.

(F) Representative immunofluorescence of a human thymus entire paraffin section (5 mm) analyzed for multiplex multispectral imaging (AKOYA): KRT15 (white), BCAM (green), and TP63 (red). Nuclei counterstained with DAPI. Scale bars, 1 mm (n = 6, human thymi).

(G) Higher magnification of subcapsular and medullary areas show rare cells with triple colocalization of KRT15, BCAM, and TP63. Scale bars, 50 μm. BCAM-single-positive endothelium is marked by asterisks (^∗). Third panel of each area highlights BCAM^posTP63^pos single cells in the subcapsular (left) and medullary areas (right). Scale bars, 20 μm.

(H) Triple-positive (KRT15^pos, BCAM^pos, and TP63^pos) single cells have been quantified as both absolute number per annotated area and percentage of total epithelial cells (TP63^pos) in each cortical and medullary area (10–15 cortical and medullary annotated areas, average annotation size 0.29 mm², n= 6 human thymi). Medulla shows a higher number of triple-positive cells than cortex. Significance: Mann-Whitney test, non-parametric; ^∗∗∗∗p < 0.0001.

(I) Triple-positive cells were quantified as both absolute number per annotated area and percentage of total epithelial cells (TP63^pos) in CC and SCap areas. SCap areas are enriched for triple-positive cells. Significance: Mann-Whitney test, non-parametric; ^∗∗∗∗p < 0.0001.