Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments

Background: The thymus, responsible for T cell-mediated adaptive immune system, has a structural and functional complexity that is not yet fully understood. Until now, thymic anatomy has been studied using histological thin sections or confocal microscopy 3D reconstruction, necessarily for limited volumes. Methods: We used Phase Contrast X-Ray Computed Tomography to address the lack of whole-organ volumetric information on the microarchitecture of its structural components. We scanned 15 human thymi (9 foetal and 6 postnatal) with synchrotron radiation, and repeated scans using a conventional laboratory x-ray system. We used histology, immunofluorescence and flow cytometry to validate the x-ray findings. Results: Application to human thymi at pre- and post-natal stages allowed reliable tracking and quantification of the evolution of parameters such as size and distribution of Hassall's Bodies and medulla-to-cortex ratio, whose changes reflect adaptation of thymic activity. We show that Hassall's bodies can occupy 25% of the medulla volume, indicating they should be considered a third thymic compartment with possible implications on their role. Moreover, we demonstrate compatible results can be obtained with standard laboratory-based x-ray equipment, making this research tool accessible to a wider community. Conclusions: Our study allows overcoming the resolution and/or volumetric limitations of existing approaches for the study of thymic disfunction in congenital and acquired disorders affecting the adaptive immune system.