Three-Dimensional Visualization of the Mouse Thymus Organization in Health and Immunodeficiency

Abstract

Lymphoid organs exhibit complex structures tightly related to their function. Surprisingly, although the thymic medulla constitutes a specialized microenvironment dedicated to the induction of T cell tolerance, its three-dimensional topology remains largely elusive because it has been studied mainly in two dimensions using thymic sections. To overcome this limitation, we have developed an automated method for full organ reconstruction in three dimensions, allowing visualization of intact mouse lymphoid organs from a collection of immunolabeled slices. We validated full organ reconstruction in three dimensions by reconstructing the well-characterized structure of skin-draining lymph nodes, before revisiting the complex and poorly described corticomedullary organization of the thymus. Wild-type thymi contain ∼200 small medullae that are connected to or separated from a major medullary compartment. In contrast, thymi of immunodeficient Rag2−/− mice exhibit only ∼20 small, unconnected medullary islets. Upon total body irradiation, medullary complexity was partially reduced and then recovered upon bone marrow transplantation. This intricate topology presents fractal properties, resulting in a considerable corticomedullary area. This feature ensures short distances between cortex and medulla, hence efficient thymocyte migration, as assessed by mathematical models. Remarkably, this junction is enriched, particularly in neonates, in medullary thymic epithelial cells expressing the autoimmune regulator. The emergence of a major medullary compartment is induced by CD4+ thymocytes via CD80/86 and lymphotoxin-α signals. This comprehensive three-dimensional view of the medulla emphasizes a complex topology favoring efficient interactions between developing T cells and autoimmune regulator–positive medullary thymic epithelial cells, a key process for central tolerance induction.

Tissue-Specific Distribution of iNKT Cells Impacts Their Cytokine Response

“…We found medullary frequencies of iNKT cells averaged 2.7% in C57BL/6 (B6) mice and 5.6% in BALB/c mice, whereas in the cortex, they were 0.27% and 0.32% respectively (Supplementary Figure 2D). Although the medullary frequency of iNKT cells was about 10 times higher than that of cortex, the thymic cortex occupies, on average, approximately 80% of the cut section (Irla et al, 2013b) and its cellularity is 1.7 fold higher than that of the medulla (data not shown). Correcting for this, we estimate that on average 30% of all iNKT cells were in the thymic cortex regardless of subset or mouse strain (Figure 2B), in contrast to most mature cell populations that primarily reside in the medulla.…”

Computational Approach to 3D Modeling of the Lymph Node Geometry

“…In the homeostatic case, which we consider, the B cell follicles are rather stable in forming spheroid-type shapes. The basic starting structure of the B cell follicles is defined a priori using original data quantitatively consistent with an independent study [20]. By marking several points along the B cell follicle, we created a piecewise-linear approximation of the B cell follicle border as shown in Figure 3a.…”

“…The blood vessels and high endothelial venules (HEVs) in the LN were approximated using the jogged line approach as described above and then triangulated (Figure 4). In general, the appearance and localization of the trabeculae, blood vessels and cortical and medullar sinuses is highly variable between different LNs [16,20]. The constructions of trabeculae, blood vessels and medullar sinuses are based on our original 3D confocal data including those from [17].…”

“…The noise was used to make the appearance of the SCS closer to a realistic LN. Indeed, the boundary of the SCS, as shown in imaging-based reconstructions [20], has a different appearance than an ideal sphere. It is rather an irregular surface with mathematically unknown yet pattern properties.…”

“…Three major compartments of a mesenteric rat LN-sinus, paracortex and follicles-were 3D reconstructed in [18,19] for simulations of T cell transit through the LN. Recent 3D reconstruction and quantification of macro-structures such as B cell follicles, T cell zone and subcapsular sinus of an inguinal mouse LN from sections stained with immunofluorescence antibodies is based on original software (FOR3D) implemented in MATLAB [20].…”