Thymic compartmentalization and maturation of mTECs, DCs, and nTregs is abrogated in patients with severe defects in T-cell development but is preserved in a patient whose genetic defects are largely permissive for T-cell development. Detailed analysis of the thymic biopsy from a representative normal thymus (A) shows defined CMD (hematoxylin and eosin staining) with normal compartmentalization of CK8⁺CK5⁻ cTECs (CK8, red staining) and CK8⁻CK5⁺ mTECs (CK5, green staining). Mature mTECs express Cld4 and Aire (brown staining, top and bottom images, respectively). In contrast, mutations that abrogate T-cell development (reticular dysgenesis, B; γc-null SCIDX1, C; RAG2-null T⁻B⁻SCID, D) or that are only partially permissive for T-cell development (Omenn syndrome associated with RMRP mutations, E), are associated with profound atrophy and loss of CMD (hematoxylin and eosin staining), highlighted by the presence of a diffuse epithelial network mostly composed of CK5 and CK8 double-positive immature TECs (yellow staining). No expression of claudin-4 (Cld4) and Aire was detected in these samples. In contrast, as shown in panel F, the biopsy from the patient carrying a hypomorphic R222C mutation in the IL2RG gene, permissive for T-cell development, shows normal thymic architecture with CMD (hematoxylin and eosin staining), normal distribution of CK8⁺CK5⁻ cTECs (CK8, red) and CK8⁻CK5⁺ mTECs (CK5, green), and expression of Cld4 and Aire (brown staining, top and bottom images, respectively). In the normal thymus (A), CD11c⁺ (top image, red staining) and S-100⁺ (bottom image, brown staining) DCs are distributed in the medullary areas. Combined CD11c and CD163 staining differentially marks CD11c⁺ DCs in the medullary region and CD163⁺ macrophages, which are primarily distributed into the cortex, with only rare CD11c and CD163 double-positive cells. Conversely, severe depletion of DCs is present in the thymic biopsies of all patients whose genetic defects severely compromise T-cell development. (B-E) Absence of S-100⁺ cells in all patients. In addition, CD11c⁺cells, although present in good number, largely coexpress CD163, indicating a macrophage phenotype. Rare CD11c⁺CD163⁻ DCs have been observed in the patient with Omenn syndrome (E). In the control thymus, Foxp3⁺ nTreg clusters around mature activated CD208⁺ (DC-LAMP) DCs, as highlighted by double-staining procedures (A). In contrast, thymic biopsies from patients with genetic defects that are nonpermissive for T-cell development show absence of mature activated CD208⁺ (DC-LAMP) DCs and Foxp3⁺ nTregs (B-D). The thymic biopsy from the Omenn syndrome patient (E) shows absence of CD208⁺ (DC-LAMP) DCs but focal expression of rare Foxp3⁺ cells (E). In contrast, the thymus from the patient with hypomorphic R222C mutation in the IL2RG gene demonstrates normal distribution of both S-100⁺ and CD11c⁺ DCs (F, CD11c⁺ top image, red staining; S-100⁺ bottom image, brown staining) along with the evidence of Foxp3⁺ nTreg interacting with mature activated CD208⁺ (DC-LAMP) DCs (F). Hematoxylin and eosin staining, original magnification ×10; immunofluorescence stainings, original magnification ×20: CK5 (green), CK8 (red), nuclei (blue), merge (yellow). Single immunohistochemical stainings, original magnification ×40: Cld4, Aire, S-100 (brown) and CD11c (red); double immunohistochemical stainings, original magnification ×40: CD11c (blue) and CD163 (brown); Foxp3 (blue) and DCLAMP (CD208; brown).