Histological Analysis

Following sacrifice, thymus and gut tissue isolated from mice were formalin-fixed and paraffin-embedded. Hematoxylin and Eosin (H&E) staining was used to assay basic histopathological changes. Paraffin sections were de-waxed, rehydrated, endogenous peroxidase activity was blocked by 0.1% H₂O₂, and nonspecific background reduced with Rodent Block M (Biocare Medical, Concord, CA, USA) before heat-based antigen-retrieval treatment and incubation with antibodies. Depending on the primary antibodies used, sections were incubated with Rat-on-Mouse HRP-Polymer (Biocare Medical) or MACH 1™ Universal HRP Polymer Kit (Biocare Medical) or 4plus Streptavidin HRP label (Biocare Medical); reactions were developed in Biocare's Betazoid DAB and nuclei counterstained with Hematoxylin. Digital images were acquired with an Olympus XC50 camera mounted on a BX51 microscope (Olympus, Tokyo, Japan), with CellF Imaging software (Soft Imaging System GmbH, Münster, Germany) or with a Nikon Eclipse E600 Microscope (Nikon) with NIS Elements Software (Nikon). The following primary antibodies were used on thymic sections: rabbit anti-cytokeratin 5 (CK5) (1:200; Covance), rat anti-cytokeratin 8 (CK8) (1:200; Developmental Studies Hybridoma Bank); rat anti-mouse AIRE (1:300; Millipore); rat anti-mouse FOXP3 (1:100; eBioscience); biotin-UEA (Biotinylated Ulex Europaeus Agglutinin I) (1:800; Vector Laboratories). Rabbit anti-CD3 primary antibody (1:100; ThermoFisher Scientific) was used on gut sections.

To quantify abnormalities of colon pathology, a histological score was used. The degree of colic alterations was blindly graded using combined scores including: the grade of inflammation (grade 0, no evidence of inflammation; grade 1, low; grade 2, moderate; grade 3, high; grade 4, intense and diffuse level of inflammation), the structural changes of intestinal layers (grade 0, absence; grade 1, focal; grade 2, partial; grade 3, diffuse level of structural changes), and the gland secretion alterations (grade 0, normal; grade 1, slight gland secretion alterations; grade 2, moderate; grade 3, diffuse gland secretion alterations). The cumulative total scores ranged from 0 to 10.

Human thymic tissue sample was formalin-fixed and paraffin-embedded. Sections were used for routine H&E staining and treated as described above. Nonspecific background was reduced with Background Sniper (Biocare Medical, Concord, CA, USA) before heat-based antigen-retrieval treatment and incubated with primary antibodies. The following primary antibodies were used: rat anti-human FOXP3 (1:100; eBioscience), mouse anti-human AIRE (1:3000; kindly provided by Prof P. Peterson, University of Tartu, Tartu, Estonia), biotin-UEA (Biotinylated Ulex Europaeus Agglutinin I) (1:800; Vector Laboratories) and Claudin-4 (used according to local standards of Pathological Anatomy Unit of Spedali Civili, Brescia, Italy). Sections were processed as described above for the murine experiments. Morphometric analysis was performed using Olympus Slide Scanner VS120-L100 (Olympus, Tokyo, Japan) and Image-pro software (Olympus) was used to analyze them. Digital images were acquired with the same instruments and software as described above.