Alteration in Three-Dimensional Microenvironment of Themis-deficient Murine Thymus Using Image Cytometry Approach

Abstract

Abstract Themis is a T-cell intrinsic protein involved in selection during thymocyte development in the thymus. Thus, Themis-deficient mice have gravely reduced numbers of T-cells. Despite extensive research on thymocyte signalling, the exact role of Themis remains a mystery. As the thymic 3-dimensional (3D) microenvironment is a critical element in supporting thymocyte development and vice versa, we were interested to investigate the effect of Themis deficiency on thymic components and processes. In this study, we paired 3D imaging with conventional flow cytometry to understand thymic development in normal and Themis-deficient condition. Current conventional 2-dimensional (2D) histology and 3D reconstruction from 2D sections are unsatisfactory for studying thymus morphology. We thus established a novel approach for whole thymus 3D imaging to visualize developing thymocytes and supporting stroma, such as epithelial cells, myeloid cells and vasculature. 3D imaging coupled with quantitative analysis of images using image cytometry allows for objective comparison of the thymic microenvironment between sample groups. Here, we show an altered medullary and vasculature organization in Themis−/− thymus, suggesting a suboptimal 3D niche supporting thymocyte development. Correspondingly, mature single-positive thymocytes which are dependent on the medullary environment are altered phenotypically. In addition, we detected higher levels of apoptosis in Themis−/− cortex and an increase in clonally deleted double-positive thymocytes. Our findings demonstrate how Themis-deficiency affects developing thymocytes and its supporting stromal components.