Tissue morphometry

H&E, pan-cytokeratin (CK) (AE1/AE3), and CD1a immunohistochemical slides were digitally scanned at × 40 magnification using a Leica SCN slide scanner (Leica Microsystems), and examined by a pathologist (LPH) using virtual microscopy (ImageScope software; Leica Biosystems). The total area of thymus tissue on the slide (“total area”) as defined by H&E stain, the area containing thymic epithelium (“TE area”, as defined by CK immunohistochemistry), and the area containing immature thymocytes (“cortical area”, as defined by CD1a immunohistochemistry) were determined by outlining them using the “pen tool” provided by the ImageScope software, as described previously [21] (Fig. S1). Individual areas identified for each tissue (in μm²) were imported into Microsoft Excel for summation and then rounded to the nearest mm² for data presentation. To adjust for differences in amounts of thymus tissue on each slide, TE, and cortical areas were converted to % of the total area by dividing by the total area of thymus tissue examined. Tissue content of developing thymocytes was defined by the % cortical area, i.e., the percentage of the tissue that contained CD1a⁺ immature thymocytes.